Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

Eva Doleželová,Alena Zíková,Luke Guddat,Tomáš Klejch,Petr Špaček,Dana Hocková,Martina Slapničková

doi:10.1038/s41598-021-91747-6

Abstract

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

Highlights

Trypanosomatid parasites (e.g. Trypanosoma spp., Leishmania spp.) are incapable of purine synthesis de novo and acquire purine derivates from their e nvironment[1]
Upon its entry to the cell, the adenosine can be converted to the adenosine monophosphate (AMP) by an adenosine kinase (AK)[19,20] or it can be cleaved to the adenine by a nucleoside hydrolase (NH)[21] and be converted to the AMP by an adenine phosphoribosyltransferase (APRT)[22]
In T. brucei bloodstream form (BSF) parasites, the APRT1 is localized in the cytosol and the APRT2 shows a partial distribution between the cytosolic compartment and glycosomes

Summary

Introduction

Trypanosomatid parasites (e.g. Trypanosoma spp., Leishmania spp.) are incapable of purine synthesis de novo and acquire purine derivates from their e nvironment[1]. Acyclic nucleoside phosphonates (ANPs) represent a group of compounds whose biological activity is based on their structural resemblance to the natural n ucleotides[8,9] Their flexibility enables them to adopt a conformation suitable for the interaction with the active site of the nucleotide binding enzymes. We determined the crystal structures of two T. brucei PSP enzymes, 6-oxopurine PRTases, in the complex with several ANPs, and showed that the prodrugs of the selected inhibitors possess strong anti-trypanosomal activity in the cell-based assay[6,17,18] Inspired by these results, here we focused on the 6-aminopurine salvage route, which is linked to the 6-oxopurine pathway via an AMP deaminase that converts AMP to IMP (Fig. 1). Their simultaneous deletion did not affect the growth of the PCF cells in vitro[22]

Methods

Results

Conclusion